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July 7, 2019  |  

Full-genome sequence of Listeria monocytogenes strain H34, isolated from a newborn with sepsis in Uruguay.

The foodborne pathogen Listeria monocytogenes causes severe disease mainly in the vulnerable populations of the young, old, pregnant, and immunocompromised. Here, we present the genome sequence of L. monocytogenes H34, a serotype 1/2b, lineage I, sequence type 489 (ST489) strain, isolated from a neonatal sepsis case in Uruguay. Copyright © 2017 Muchaamba et al.


July 7, 2019  |  

Benzalkonium tolerance genes and outcome in Listeria monocytogenes meningitis.

Listeria monocytogenes is a food-borne pathogen that can cause meningitis. The listerial genotype ST6 has been linked to increasing rates of unfavourable outcome over time. We investigated listerial genetic variation and the relation with clinical outcome in meningitis.We sequenced 96 isolates from adults with listerial meningitis included in two prospective nationwide cohort studies by whole genome sequencing, and evaluated associations between bacterial genetic variation and clinical outcome. We validated these results by screening listerial genotypes of 445 cerebrospinal fluid and blood isolates from patients over a 30-year period from the Dutch national surveillance cohort.We identified a bacteriophage, phiLMST6 co-occurring with a novel plasmid, pLMST6, in ST6 isolates to be associated with unfavourable outcome in patients (p 2.83e-05). The plasmid carries a benzalkonium chloride tolerance gene, emrC, conferring decreased susceptibility to disinfectants used in the food-processing industry. Isolates harbouring emrC were growth inhibited at higher levels of benzalkonium chloride (median 60 mg/L versus 15 mg/L; p <0.001), and had higher MICs for amoxicillin and gentamicin compared with isolates without emrC (both p <0.001). Transformation of pLMST6 into naive strains led to benzalkonium chloride tolerance and higher MICs for gentamicin.These results show that a novel plasmid, carrying the efflux transporter emrC, is associated with increased incidence of ST6 listerial meningitis in the Netherlands. Suggesting increased disease severity, our findings warrant consideration of disinfectants used in the food-processing industry that select for resistance mechanisms and may, inadvertently, lead to increased risk of poor disease outcome. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.


July 7, 2019  |  

Complete genome sequences of two Salmonella enterica subsp. enterica serovar Enteritidis strains isolated from egg products in the United States.

Egg-associated salmonellosis is an important public health problem in many countries. Here, we report the genome sequences, including plasmids, of two strains of Salmonella enterica subsp. enterica serovar Enteritidis isolated from egg products in 2012 and 2013 in the United States. This will provide more information and insight into the research about egg-associated salmonellosis. Copyright © 2017 Hu et al.


July 7, 2019  |  

2015 epidemic of severe Streptococcus agalactiae sequence type 283 infections in Singapore associated with the consumption of raw freshwater fish: a detailed analysis of clinical, epidemiological, and bacterial sequencing data.

Streptococcus agalactiae (group B Streptococcus [GBS]) has not been described as a foodborne pathogen. However, in 2015, a large outbreak of severe invasive sequence type (ST) 283 GBS infections in adults epidemiologically linked to the consumption of raw freshwater fish occurred in Singapore. We attempted to determine the scale of the outbreak, define the clinical spectrum of disease, and link the outbreak to contaminated fish.Time-series analysis was performed on microbiology laboratory data. Food handlers and fishmongers were screened for enteric carriage of GBS. A retrospective cohort study was conducted to assess differences in demographic and clinical characteristics of patients with invasive ST283 and non-ST283 infections. Whole-genome sequencing was performed on human and fish ST283 isolates from Singapore, Thailand, and Hong Kong.The outbreak was estimated to have started in late January 2015. Within the study cohort of 408 patients, ST283 accounted for 35.8% of cases. Patients with ST283 infection were younger and had fewer comorbidities but were more likely to develop meningoencephalitis, septic arthritis, and spinal infection. Of 82 food handlers and fishmongers screened, none carried ST283. Culture of 43 fish samples yielded 13 ST283-positive samples. Phylogenomic analysis of 161 ST283 isolates from humans and fish revealed they formed a tight clade distinguished by 93 single-nucleotide polymorphisms.ST283 is a zoonotic GBS clone associated with farmed freshwater fish, capable of causing severe disease in humans. It caused a large foodborne outbreak in Singapore and poses both a regional and potentially more widespread threat.


July 7, 2019  |  

Comparative genomic analysis reveals genetic features related to the virulence of Bacillus cereus FORC_013.

Bacillus cereus is well known as a gastrointestinal pathogen that causes food-borne illness. In the present study, we sequenced the complete genome of B. cereus FORC_013 isolated from fried eel in South Korea. To extend our understanding of the genomic characteristics of FORC_013, we conducted a comparative analysis with the published genomes of other B. cereus strains.We fully assembled the single circular chromosome (5,418,913 bp) and one plasmid (259,749 bp); 5511 open reading frames (ORFs) and 283 ORFs were predicted for the chromosome and plasmid, respectively. Moreover, we detected that the enterotoxin (NHE, HBL, CytK) induces food-borne illness with diarrheal symptom, and that the pleiotropic regulator, along with other virulence factors, plays a role in surviving and biofilm formation. Through comparative analysis using the complete genome sequence of B. cereus FORC_013, we identified both positively selected genes related to virulence regulation and 224 strain-specific genes of FORC_013.Through genome analysis of B. cereus FORC_013, we identified multiple virulence factors that may contribute to pathogenicity. These results will provide insight into further studies regarding B. cereus pathogenesis mechanism at the genomic level.


July 7, 2019  |  

Whole-genome comparative analysis of Salmonella enterica serovar Newport strains reveals lineage-specific divergence.

Salmonella enterica subsp. enterica serovar Newport has been associated with various foodborne outbreaks in humans and animals. Phylogenetically, serovar Newport is one of several Salmonella serovars that are polyphyletic. To understand more about the polyphyletic nature of this serovar, six food, environment, and human isolates from different Newport lineages were selected for genome comparison analyses. Whole genome comparisons demonstrated that heterogeneity mostly occurred in the prophage regions. Lineage-specific characteristics were also present in the Salmonella pathogenicity islands and fimbrial operons. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.


July 7, 2019  |  

Comparative sequence analysis of multidrug-resistant IncA/C plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.


July 7, 2019  |  

IS26-mediated formation of a virulence and resistance plasmid in Salmonella Enteritidis.

To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380.The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis.Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility.This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.


July 7, 2019  |  

Benchmark datasets for phylogenomic pipeline validation, applications for foodborne pathogen surveillance.

As next generation sequence technology has advanced, there have been parallel advances in genome-scale analysis programs for determining evolutionary relationships as proxies for epidemiological relationship in public health. Most new programs skip traditional steps of ortholog determination and multi-gene alignment, instead identifying variants across a set of genomes, then summarizing results in a matrix of single-nucleotide polymorphisms or alleles for standard phylogenetic analysis. However, public health authorities need to document the performance of these methods with appropriate and comprehensive datasets so they can be validated for specific purposes, e.g., outbreak surveillance. Here we propose a set of benchmark datasets to be used for comparison and validation of phylogenomic pipelines.We identified four well-documented foodborne pathogen events in which the epidemiology was concordant with routine phylogenomic analyses (reference-based SNP and wgMLST approaches). These are ideal benchmark datasets, as the trees, WGS data, and epidemiological data for each are all in agreement. We have placed these sequence data, sample metadata, and “known” phylogenetic trees in publicly-accessible databases and developed a standard descriptive spreadsheet format describing each dataset. To facilitate easy downloading of these benchmarks, we developed an automated script that uses the standard descriptive spreadsheet format.Our “outbreak” benchmark datasets represent the four major foodborne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) and one simulated dataset where the “known tree” can be accurately called the “true tree”. The downloading script and associated table files are available on GitHub: https://github.com/WGS-standards-and-analysis/datasets.These five benchmark datasets will help standardize comparison of current and future phylogenomic pipelines, and facilitate important cross-institutional collaborations. Our work is part of a global effort to provide collaborative infrastructure for sequence data and analytic tools-we welcome additional benchmark datasets in our recommended format, and, if relevant, we will add these on our GitHub site. Together, these datasets, dataset format, and the underlying GitHub infrastructure present a recommended path for worldwide standardization of phylogenomic pipelines.


July 7, 2019  |  

Complete genome sequence of Staphylococcus epidermidis ATCC 12228 chromosome and plasmids, generated by long-read sequencing.

Staphylococcus epidermidis ATCC 12228 was sequenced using a long-read method to generate a complete genome sequence, including some plasmid sequences. Some differences from the previously generated short-read sequence of this nonpathogenic and non-biofilm-forming strain were noted. The assembly size was 2,570,371 bp with a total G+C% content of 32.08%. Copyright © 2017 MacLea and Trachtenberg.


July 7, 2019  |  

Parallel evolution of two clades of a major Atlantic endemic Vibrio parahaemolyticus pathogen lineage by independent acquisition of related pathogenicity islands.

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States (US). This change coincided with a bacterial population shift towards human pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43 and ST636) to near-shore areas off the Atlantic coast of the Northeast US. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins, or contained a chromosome I-inserted island we call VPaIß that encodes a type three secretion system (T3SS2ß) typical of Trh hemolysin-producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI? that encodes both tdh and trh and that was inserted in chromosome II. VPaI? was derived from VPaIß but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI?-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages. that collectively cause 67% of Northeast US infections as of 2016.IMPORTANCE The availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast US coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also to become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI? along the US Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

Genomic insights into the pathogenicity and environmental adaptability of Enterococcus hirae R17 isolated from pork offered for retail sale.

Genetic information about Enterococcus hirae is limited, a feature that has compromised our understanding of these clinically challenging bacteria. In this study, comparative analysis was performed of E. hirae R17, a daptomycin-resistant strain isolated from pork purchased from a retail market in Beijing, China, and three other enterococcal genomes (Enterococcus faecium DO, Enterococcus faecalis V583, and E. hirae ATCC™ 9790). Some 1,412 genes were identified that represented the core genome together with an additional 139 genes that were specific to E. hirae R17. The functions of these R17 strain-specific coding sequences relate to the COGs categories of carbohydrate transport and metabolism and transcription, a finding that suggests the carbohydrate utilization capacity of E. hirae R17 may be more extensive when compared with the other three bacterial species (spp.). Analysis of genomic islands and virulence genes highlighted the potential that horizontal gene transfer played as a contributor of variations in pathogenicity in this isolate. Drug-resistance gene prediction and antibiotic susceptibility testing indicated E. hirae R17 was resistant to several antimicrobial compounds, including bacitracin, ciprofloxacin, daptomycin, erythromycin, and tetracycline, thereby limiting chemotherapeutic treatment options. Further, tolerance to biocides and metals may confer a phenotype that facilitates the survival and adaptation of this isolate against food preservatives, disinfectants, and antibacterial coatings. The genomic plasticity, mediated by IS elements, transposases, and tandem repeats, identified in the E. hirae R17 genome may support adaptation to new environmental niches, such as those that are found in hospitalized patients. A predicted transmissible plasmid, pRZ1, was found to carry several antimicrobial determinants, along with some predicted pathogenic genes. These data supported the previously determined phenotype confirming that the foodborne E. hirae R17 is a multidrug-resistant pathogenic bacterium with evident genome plasticity and environmental adaptability.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


July 7, 2019  |  

Complete genome sequence of the pathogenic Vibrio vulnificus type strain ATCC 27562.

Vibrio vulnificus has the highest death rate and economic burden per case of any foodborne pathogen in the United States. A complete genome sequence of the type strain promotes comparative analyses with other clinical and environmental isolates, improving our understanding of this important human pathogen and successful environmental organism. Copyright © 2017 Rusch and Rowe-Magnus.


July 7, 2019  |  

Spontaneous loss of virulence in natural populations of Listeria monocytogenes.

The pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA(-)/LLO(-)) mutants belonged to phylogenetically diverse clades of L. monocytogenes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA(-)/LLO(-) mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen. Copyright © 2017 Maury et al.


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